Fluorescent Labels and Dyes catalogue 2007/2008
Cover: Actin (PtK2 - Male Rat Kangaroo Kidney Epithelial Cells) ATTO 532 labeled phalloidin bound to actin.
Fluorescent Labels and Dyes catalogue 2007/2008
Contents ATTO-TEC - The Company
6
Contact Information
7
Introduction Fluorescence How to Choose the Right Label Fluorescence Resonance Energy Transfer (FRET) Table of Förster-radii for ATTO-dyes Properties of Fluorescent Labels Structure of Fluorescent Labels Reactive Labels and Conjugates About this Catalogue
8 8 8 10 12 14 16 17 19
Fluorescent Labels
20
ATTO 390 ATTO 425 ATTO 465 ATTO 488 ATTO 495 ATTO 520 ATTO 532 ATTO 550 ATTO 565 ATTO 590 ATTO 594 ATTO 610 ATTO 611X ATTO 620 ATTO 633 ATTO 635 ATTO 637 ATTO 647 ATTO 647N ATTO 655 ATTO 680
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
4
ATTO 700 ATTO 725 ATTO 740
43 44 45
Fluorescence Quenchers
46
ATTO 540Q ATTO 580Q ATTO 612Q
47 48 49
Large Stokes-Shift Dyes ATTO 390, ATTO 425, ATTO 465, ATTO 611X
50 51
Customized Dyes and Services
52
Labeling Procedures
56
Labeled Nucleotides
64
Adenosine derived nucleotides Cytidine derived nucleotides Guanosine derived nucleotides Uridine derived nucleotides
64 66 67 68
Synthesis and Labeling of DNA
70
Picture Gallery
71
List of Abbreviations
74
Acknowledgements
75
5
ATTO-TEC Fluorescence is Our Business
ATTO-TEC Fluorescence is Our Business
ATTO-TEC GmbH Fluorescence - Our Passion Although the phenomenon as such has been known for more than a century, it was only during the last few decades that fluorescence has developed into a powerful tool in biochemistry and medical diagnostics. Applications now have become so diversified and sophisticated that there is an ever growing demand for new and better fluorescent dyes. To take up the challenge ATTO-TEC GmbH was founded in 1999. The company has grown continually since. It is staffed by internationally renowned scientists with long-time expertise in dye chemistry and physics. Consequently ATTO-dyes are used now with great success by scientists throughout the world. Researchers prefer ATTO-products for their high purity and excellent performance. In many applications ATTO-dyes are not merely an alternative, they are the better choice. We are proud to present to you the new edition of our catalogue. In this booklet you will find many new and innovative fluorescent labels - proprietary compounds covered by ATTO-TEC patents and patent applications. – Our continuous research is aimed at optimum dye solutions for our customers. It is a pleasure to introduce you to ATTO-TEC – the company that creates success with fluorescent dyes. The Team of ATTO-TEC
Business Address: ATTO-TEC GmbH Am Eichenhang 50 D-57076 Siegen Germany
HUVEC: alpha-Tubulin/ATTO 532; E-Cadherin/ATTO 655 and DAPI
Mail Address: ATTO-TEC GmbH P.O. Box 10 08 64 D-57008 Siegen Germany Phone: Fax: E-mail: http:
+49(0)-271-2 38 53-0 +49(0)-271-2 38 53-11
[email protected] www.atto-tec.com
HUVEC: Inhibitor apoptosis protein/ ATTO 550; E-Cadherin/ATTO 655 and DAPI
To Place Orders: Phone: Fax: E-mail: http:
+49(0)-271-2 38 53-0 +49(0)-271-2 38 53-11
[email protected] www.atto-tec.com
HUVEC: Vimentin/ATTO 532; E-Cadherin/ATTO 655 and DAPI
Headquarters of ATTO-TEC GmbH
6
7
Introduction
Fluorescence The emission of light by molecules, so-called fluorescence, has been known for more than one hundred years. However, it was only during the last few decades that versatile light sources (lasers etc.) and highly sensitive detectors have been developed. In recent years fluorescence spectroscopy has become a powerful tool with outstanding sensitivity. By sophisticated techniques nowadays even single molecules can be studied via fluorescence. Most molecules of interest, e.g. in biochemistry, do not show fluorescence of their own. However, they may be chemically connected, i.e. labeled, with a fluorescent dye. Therefore the development of dyes that are suitable as labels is a subject of great importance in modern biology, medicine and diagnostics.
Introduction
between excitation wavelength and fluorescence (the latter being independent of excitation wavelength with all dyes) provides the additional advantage of better discrimination against scattered excitation light. The table below provides an overview of some frequently used excitation sources and recommended ATTO-labels.
Light source
Emission line
Best suited dyes
Mercury arc lamp
365 nm
ATTO 390
405 nm
ATTO 425
436 nm
ATTO 425, ATTO 465
546 nm
ATTO 550, ATTO 565
577 nm
ATTO 590, ATTO 594, ATTO 610, ATTO 611X
488 nm
ATTO 488, ATTO 520 ATTO
514 nm
520, ATTO 532, ATTO 550
Nd:YAG laser
532 nm
ATTO 532, ATTO 550, ATTO 565
He-Ne laser
633 nm
ATTO 633, ATTO 635, ATTO 637, ATTO 647, ATTO 647N
Krypton-Ion laser
647 nm
ATTO 647, ATTO 647N, ATTO 655, ATTO 680
676 nm
ATTO 680, ATTO 700, ATTO 725, ATTO 740
635 nm
ATTO 635, ATTO 637, ATTO 647, ATTO 647N, ATTO 655
How to Choose the Right Label To obtain the best possible results several factors have to be considered. First is the source of excitation: To reduce interference due to autofluorescence of the sample an excitation wavelength above 550 nm or even 600 nm is advisable. Secondly the label should show strong absorption at the excitation wavelength as well as high fluorescence quantum yield. Finally the emission spectrum of the label should match the transmission of the applied filter set. The filter set, in turn, must be chosen such that it rejects the excitation light scattered by the sample, yet transmits the fluorescence as effectively as possible. For example, when using a diode laser of wavelength 635 nm as the excitation source and a custom made filter set with high transmittance between 650 nm and 750 nm, ATTO 647N would be a very good choice. As can be seen from the list of ATTO-labels in this catalogue, ATTO 647N (p. 40) has a high extinction coefficient at 635 nm (as follows from εmax and inspection of the absorption curve) as well as an excellent quantum yield of fluorescence (ηfl = 0.65). It is to be noted, however, that besides optical considerations other factors may be important for the choice of label, e.g. pH-dependence, solubility, photostability, size of chromophore or linker and many others. If there is no label available with an absorption maximum exactly matching the applied excitation source, a label with a slightly longer wavelength should be chosen. The absorbance will not decrease drastically, but the larger shift 8
Argon-Ion laser
Diode laser
9
Introduction
Introduction
Fluorescence Resonance Energy Transfer (FRET) FRET is becoming more and more important as a method to determine distances at the molecular level and to study dynamic processes like binding of antibody/antigen pairs. If two dye molecules are located close to each other, their transition dipoles can interact, and energy can be transferred from one dye molecule to the other. The rate of energy transfer kET is in good approximation given by (Förster theory):
As can be seen from the formula, the rate of energy transfer decreases with the 6th power of the distance between the dye molecules. Thus FRET is very efficient only when donor and acceptor are in close proximity. It becomes negligibly small at distances above 10 nm. Furthermore its rate is proportional to the extinction coefficient of the acceptor dye in the wavelength range of the donor fluorescence (overlap integral): FRET is most efficient, if there is a good spectral overlap between fluorescence of donor and absorption of acceptor. A practical measure of FRET efficiency is the distance at which the rate kET of energy transfer equals the rate of donor fluorescence. This so-called Försterradius R0 is given by:
∞
k ET
NA n τ0 r F(λ)
9 ln10 κ2 = ⋅ F(λ ) ⋅ ε(λ ) ⋅ λ 4 dλ 5 4 6 ∫ 128π N A n τ0 r 0
ε(λ)
Avogadro constant index of refraction radiative decay time of donor distance between donor and acceptor molecule fluorescence spectrum of donor, normalized according ∫F(λ) dλ = 1 extinction coefficient of acceptor
κ2 =
(cosφDA – 3 cosφD cosφA)2 φDA φD φA
10
angle between transition dipoles of donor and ac ceptor angle between donor transition dipole and line connecting the dipoles angle between acceptor transition dipole and line connecting the dipoles
∞
9 ln10 κ 2 ηfl 4 ⋅ λ ⋅ ε λ ⋅ λ R = F( ) ( ) dλ 5 4 ∫ 128π N A n 0 6 0
ηfl τfl
fluorescence quantum yield of donor, ηfl = τfl / τ0 fluorescence decay time of donor
A table of Förster-radii for ATTO-dyes is presented on p. 12-13. These values have been calculated with the assumption of statistical orientation of both donor and acceptor (orientation factor κ2 = 2/3), a situation typically encountered in solutions of unbound dye molecules. However, in case of dye labeled biomolecules the chromophores of donor and acceptor may be held rigidly in a fixed position. As a consequence the orientation factor will assume a value different from 2/3. Since for κ2 values between 0 and 4 are possible, the Förster-radius will vary accordingly. For accurate distance determinations via FRET it is important to take the relative orientation of donor and acceptor into account.
11
Introduction
Introduction
Förster-radius R0 of ATTO-dye pairs in Å (1 Å = 0.1 nm) Donor
Acceptor 611X
612Q
620
633
635
637
647
647N
655
680
700
725
740
390
40
43
41
41
39
40
39
39
40
38
35
36
36
48
425
44
47
45
45
43
44
43
43
43
41
38
36
37
52
52
465
49
52
49
49
48
49
48
48
48
46
43
41
40
60
57
57
488
53
55
53
53
52
53
51
51
50
48
44
41
40
55
56
54
54
495
51
53
51
51
50
50
49
49
49
47
44
42
41
67
64
64
61
60
520
56
59
56
55
55
56
54
53
53
50
46
43
41
68
68
67
68
66
66
532
62
64
61
61
61
62
60
59
59
57
53
50
48
58
63
69
70
68
69
550
65
67
67
66
66
67
65
65
64
62
58
55
53
61
69
72
71
73
565
69
70
69
69
69
70
69
68
68
65
61
58
56
63
66
73
590
69
71
71
73
73
73
73
74
73
71
69
66
63
62
70
594
67
68
70
73
73
73
74
75
75
74
72
69
68
64
610
64
63
66
70
71
73
72
73
76
75
74
69
68
611X
611X
44
44
45
50
52
52
57
56
62
65
66
65
64
620
620
58
64
66
65
68
70
70
69
68
67
65
633
633
60
63
62
68
69
72
73
72
72
71
635
635
54
53
58
59
63
63
62
62
61
637
637
51
56
58
61
62
62
62
61
647
647
51
52
58
60
61
61
60
647N
647N
65
72
75
74
73
72
655
655
58
64
66
66
65
680
680
59
65
67
66
700
700
58
66
66
725
725
52
56
740
740
390 425 465 488 495 520 532 550 565 590 594 610
12
390
425
465
488
495
520
532
540Q
550
565
580Q
590
594
610
14
41
50
58
59
60
56
54
53
52
47
48
45
44
36
46
59
59
61
58
56
56
55
51
51
49
35
52
51
61
56
55
56
55
53
54
50
46
61
64
63
63
63
60
41
56
56
56
57
58
57
65
66
67
57
63
54
13
Introduction
Introduction
Properties of Fluorescent Labels Apart from absorption and fluorescence there are many other dye properties that are highly relevant with respect to their suitability as labels. Most important, the dye must remain intact during irradiation. Many common labels, e.g. Fluorescein (FITC), show very low photostability. As a result sensitivity and quality of imaging are limited if high-intensity laser excitation is used and processes are to be observed over long periods of time. This is a serious draw-back with microscopy and other techniques based on the confocal principle, e.g. in single-cell detection applications. In contrast to some widely used older dyes, the new patented ATTO-labels are designed to be much more stable under prolonged irradiation.
Besides reduced background, an advantage of excitation in the red spectral region is that rugged diode lasers can be used in place of gas lasers. Diode lasers are generally less expensive and more energy-efficient. Furthermore a variety of sensitive detectors is now available for the visible-near-IR region. Excitation in the red spectral region is also advantageous when working with live cells, because damage is reduced. The fluorescence efficiency of dyes is highest in the blue and green region of the spectrum. Here the quantum yield reaches in some cases almost the theoretical limit of 100 %. Towards longer wavelengths the efficiency drops drastically, in particular so in aqueous solution. However, ATTO-TEC has been able to develop labels that show high quantum yield even around 650 nm: The new ATTO 647N fluoresces in aqueous solution twice as strong as the old Cy5TM.
ATTO 647N, 66 %
Cy5
0
10
20
30
time, min
40
50
60
Photostability of ATTO 655 compared with common Cy5TM in water. Irradiation with a 250 W tungsten-halogen lamp. Absorbance vs. time of illumination.
Many common fluorescent labels deteriorate even without any irradiation (in the dark), in particular when exposed to small concentrations of ozone present in the laboratory atmosphere. Under identical conditions of ozone exposure the new dyes ATTO 647N and ATTO 655 last up to 100 times longer than dyes like the older Cy5TM and Alexa 647TM. This is very important in microarray applications, where the dye molecules are located at the surface and thus are in direct contact with the atmosphere.
14
fluorescence intensity
absorbance
ATTO 655
Cy5, 32 %
600
700
wavelength, nm
800
Fluorescence quantum yield of ATTO 647N compared with common Cy5TM in water. Solutions of equal absorbance excited at 647 nm. Fluorescence intensity vs. wavelength.
15
Introduction
Introduction
Structure of Fluorescent Labels The molecules of most common dyes, e.g. cyanines, have a more or less flexible structure. Hence their solutions contain a mixture of several isomers with varying properties. Since the equilibrium between the isomers depends on temperature and other environmental factors, absorption and fluorescence of such dyes are ill-defined. In stark contrast to cyanines, ATTO-dyes have a molecular structure that ensures high rigidity of the chromophore. They do not form equilibria with various isomers, their optical properties are nearly independent of solvent and temperature.
•
Carbopyronin
H 3C + N CH3
N H 3C
CH3
CH3
CH3
N ATTO-labels are typically derivatives of:
•
Oxazine
H 5C 2 + N C 2H 5
•
Coumarin
H 5C 2
N
O
16
C 2H 5
ATTO-labels are designed for application in the area of life science, e.g. labeling of DNA, RNA or proteins. Characteristic features of most labels are strong absorption, high fluorescence quantum yield, excellent photostability, exceptionally high ozone resistance, and good water solubility.
Rhodamine
C 2H 5
C 2H 5
Reactive Labels and Conjugates
CO2C2H5
H 5C 2 + N
N
O
C 2H 5
•
O
O
N
C 2H 5
C 2H 5
All ATTO-labels are available as NHS-esters for coupling to amino groups and as maleimides for coupling to thiol groups. Dyes with other reactive substituents can be supplied on request. Furthermore ATTO-dyes conjugated to streptavidin and other proteins are available on request. The high affinity of streptavidin to biotin is the basis for the wide-spread use of streptavidin conjugates. In this connection all ATTO-dyes are also offered as biotin conjugates.
17
Introduction
Introduction
About this Catalogue
ATTO-dye with free COOH:
All spectral data given have been measured on aqueous solutions of the dyes with free carboxy group. When there was a tendency to aggregate, the solution was diluted sufficiently to exhibit the monomeric spectrum undisturbed by dimers. Although water is the most important solvent in biochemistry, it should be borne in mind that optical data of dyes, in particular fluorescence efficiency and decay time, depend on the solvent as well as on other environmental factors. With most ATTO-dyes this influence is very weak indeed. Furthermore optical properties depend on the derivative (free COOH, NHS-ester, etc.). In particular the fluorescence quantum yield of the maleimide may be reduced compared to the dye with free COOH. However, this is of no avail: As soon as the dye is coupled to a substrate (protein), the fluorescence is restituted.
O ATTO C OH NHS-ester:
O
O
ATTO C O
N
The spectra presented in this catalogue will help to select the dye best suited for a particular experiment. For accurate data in digitized form the reader is referred to www.atto-tec.com (Products, Documents, Spectral data). - The correction factors CF260 and CF280 aide with calculating the degree of labeling (DOL), see „Labeling Procedures“ (p. 56-61).
O maleimide:
O
The molecular weight (MW) given has the common meaning, i.e. it refers to the dye including counterions. For mass spectrometry purposes also the mass of the single-charged ion (M+ or MH+) is given.
O
ATTO C N H
N
For further details on all products as well as new developments please visit our website www.atto-tec.com.
O
biotin derivative:
O
O
ATTO C N
N
H
H
S
HN
NH O
18
19
Fluorescent Labels
Fluorescent Labels
ATTO Fluorescent Labels ATTO Fluorescence Quenchers (p. 46-49)
Label
λabs ,
εmax ,
λfl ,
ηfl ,
τfl ,
nm
M-1 cm-1
nm
%
ns
ATTO 390
390
24000
479
90
3.8
ATTO 425
436
45000
484
90
3.5
Label
λabs ,
εmax ,
nm
M-1 cm-1
Quenching Range, nm
ATTO 465
453
75000
508
55
2.2
ATTO 540Q
542
105000
500 - 565
ATTO 488
501
90000
523
80
3.2
ATTO 580Q
586
110000
535 - 610
ATTO 495
495
80000
527
45
2.4
ATTO 612Q
615
115000
555 - 640
ATTO 520
516
110000
538
90
3.8
JOE**, TET**
ATTO 532
532
115000
553
90
3.8
Alexa 532*, HEX**
ATTO 550
554
120000
576
80
3.2
TAMRA**, Cy3***
ATTO 565
563
120000
592
90
3.4
Cy3.5***, ROX**
ATTO 590
594
120000
624
80
3.7
Alexa 594*, Texas Red*
ATTO 594
601
120000
627
85
3.5
Alexa 594*
ATTO 610
615
150000
634
70
3.3
ATTO 611X
611
100000
681
35
2.5
ATTO 620
619
120000
643
50
2.9
ATTO 633
629
130000
657
64
3.2
Alexa 633*
ATTO 635
635
120000
659
25
1.9
Alexa 633*
ATTO 637
635
120000
659
25
1.9
Alexa 633*
ATTO 647
645
120000
669
20
2.3
Cy5***, Alexa 647*
ATTO 647N
644
150000
669
65
3.4
Cy5***, Alexa 647*
ATTO 655
663
125000
684
30
1.9
Cy5***, Alexa 647*
MW
molecular weight
M+
molecular weight of dye cation (HPLC-MS)
MH+
molecular weight of protonated dye (HPLC-MS)
CF260
CF260 = ε260/εmax. Correction factor used in calculation of degree of labeling (DOL) in case of dye-DNA conjugates.
CF280
CF280 = ε280/εmax. Correction factor used in calculation of degree of labeling (DOL) in case of dye-protein conjugates.
Alternative to
Alexa 488*, FITC, FAM**
ATTO 680
680
125000
700
30
1.8
Cy5.5***
ATTO 700
700
120000
719
25
1.5
Cy5.5***
ATTO 725
729
120000
752
10
0.5
ATTO 740
740
120000
764
10
0.6
Dyes with Large Stokes-Shift Label
λabs ,
εmax ,
λfl ,
ηfl ,
τfl ,
nm
M-1 cm-1
nm
%
ns
ATTO 390
390
24000
479
90
3.8
ATTO 425
436
45000
484
90
3.5
ATTO 465
453
75000
508
55
2.2
ATTO 611X
611
100000
681
35
2.5
λabs
longest-wavelength absorption maximum
εmax
molar extinction coefficient at the longest-wavelength absorption maximum
λfl
fluorescence maximum
ηfl
fluorescence quantum yield
τfl
fluorescence decay time
τ0
natural (radiative) fluorescence decay time
* Trademark of Invitrogen Corporation, ** Trademark of Applera Corporation, *** Trademark of GE Healthcare Group Companies
20
21
Fluorescent Labels
Fluorescent Labels ATTO 425
ATTO 390 Solvent: Water
Solvent: Water
λabs
=
390 nm
λabs
=
436 nm
εmax
=
2.4 x 104 M-1 cm-1
εmax
=
4.5 x 104 M-1 cm-1
λfl
=
479 nm
λfl
=
484 nm
ηfl
=
90 %
CF260 = 0.52
ηfl
=
90 %
CF260 = 0.27
τfl
=
3.8 ns
CF280 = 0.08
τfl
=
3.5 ns
CF280 = 0.23
Features: • • • • •
Features:
High fluorescence yield Large Stokes-shift Moderately hydrophilic Good solubility in polar solvents Coumarin derivative, uncharged
• • • • •
High fluorescence yield Large Stokes-shift Moderately hydrophilic Good solubility in polar solvents Coumarin derivative, uncharged
O
fluorescence
O
absorbance
O
absorbance
N
fluorescence
O N
O
O
COOH COOH 300
400
500
600
700
wavelength, nm
800
900
1000
400
500
600
700
wavelength, nm
800
900
1000
Modification
MW, g/mol
MH+, g/mol
Unit
Order Code
Modification
MW, g/mol
MH+, g/mol
Unit
Order Code
with free COOH
343.4
344
1 mg 5 mg
AD 390-21 AD 390-25
with free COOH
401.5
402
1 mg 5 mg
AD 425-21 AD 425-25
NHS-ester
440.5
441
1 mg 5 mg
AD 390-31 AD 390-35
NHS-ester
498.5
499
1 mg 5 mg
AD 425-31 AD 425-35
maleimide
465.6
466
1 mg 5 mg
AD 390-41 AD 390-45
maleimide
523.6
524
1 mg 5 mg
AD 425-41 AD 425-45
biotin derivative
653.9
654
1 mg 5 mg
AD 390-71 AD 390-75
biotin derivative
711.9
712
1 mg 5 mg
AD-425-71 AD-425-75
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
22
300
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
23
Fluorescent Labels
Fluorescent Labels ATTO 488
ATTO 465 Solvent: Water
Solvent: Water
λabs
=
453 nm
λabs
=
501 nm
εmax
=
7.5 x 104 M-1 cm-1
εmax
=
9.0 x 104 M-1 cm-1
λfl
=
508 nm
λfl
=
523 nm
ηfl
=
55 %
CF260 = 1.12
ηfl
=
80 %
CF260 = 0.25
τfl
=
2.2 ns
CF280 = 0.54
τfl
=
3.2 ns
CF280 = 0.10
Features:
ClO4
-
COOH 300
400
500
600
700
wavelength, nm
800
900
1000
300
400
500
600
700
wavelength, nm
800
900
1000
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
with free COOH
395.8
296
1 mg 5 mg
AD 465-21 AD 465-25
with free COOH
804
590
1 mg 5 mg
AD 488-21 AD 488-25
NHS-ester
492.9
393
1 mg 5 mg
AD 465-31 AD 465-35
NHS-ester
981
687
1 mg 5 mg
AD 488-31 AD 488-35
maleimide
517.9
418
1 mg 5 mg
AD 465-41 AD 465-45
maleimide
1067
712
1 mg 5 mg
AD 488-41 AD 488-45
biotin derivative
706.3
606
1 mg 5 mg
AD 465-71 AD 465-75
biotin derivative
1191
900
1 mg 5 mg
AD 488-71 AD 488-75
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
24
High fluorescence yield High photostability Very hydrophilic Excellent water solubility Very little aggregation New dye with net charge of -1
absorbance
NH2
fluorescence
+ N
H2N
• • • • • •
fluorescence
High fluorescence yield Large Stokes-shift in aqueous solution High triplet yield, intense phosphorescence in solid matrix Hydrophilic Good solubility in all polar solvents Cationic dye derived from well-known Acriflavine, perchlorate salt
absorbance
• • • • • •
Features:
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
25
Fluorescent Labels
Fluorescent Labels ATTO 520
ATTO 495 Solvent: Water
Solvent: Water
λabs
=
495 nm
λabs
=
516 nm
εmax
=
8.0 x 104 M-1 cm-1
εmax
=
1.1 x 105 M-1 cm-1
λfl
=
527 nm
λfl
=
538 nm
ηfl
=
45 %
CF260 = 0.57
ηfl
=
90 %
CF260 = 0.40
τfl
=
2.4 ns
CF280 = 0.39
τfl
=
3.8 ns
CF280 = 0.40
Features: • • • • •
Features:
High triplet yield Phosphorescent in solid matrix Moderately hydrophilic Good solubility in polar solvents Cationic dye derived from well-known Acridine Orange, perchlorate salt
• • • • • •
High fluorescence yield High thermal and photostability Moderately hydrophilic Good solubility in all polar solvents At pH > 7 reversible formation of colorless pseudobase Cationic dye closely related to well-known Rhodamine 6G, perchlorate salt
ClO4
fluorescence
H
-
N
+ H N
O ClO4
COOH 300
400
500
600
700
wavelength, nm
800
900
1000
300
400
500
600
700
wavelength, nm
800
900
-
1000
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
with free COOH
451.9
352
1 mg 5 mg
AD 495-21 AD 495-25
with free COOH
466.9
367
1 mg 5 mg
AD 520-21 AD 520-25
NHS-ester
549.0
449
1 mg 5 mg
AD 495-31 AD 495-35
NHS-ester
564.0
464
1 mg 5 mg
AD 520-31 AD 520-35
maleimide
574.0
474
1 mg 5 mg
AD 495-41 AD 495-45
maleimide
589,0
489
1 mg 5 mg
AD 520-41 AD 520-45
biotin derivative
762.4
662
1 mg 5 mg
AD 495-71 AD 495-75
biotin derivative
777.4
677
1 mg 5 mg
AD 520-71 AD 520-75
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
26
absorbance
N
absorbance
+ N
N
fluorescence
COOH
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
27
Fluorescent Labels
Fluorescent Labels ATTO 550
ATTO 532 Solvent: Water
Solvent: Water
λabs
=
532 nm
λabs
=
554 nm
εmax
=
1.15 x 105 M-1 cm-1
εmax
=
1.2 x 105 M-1 cm-1
λfl
=
553 nm
λfl
=
576 nm
ηfl
=
90 %
CF260 = 0.22
ηfl
=
80 %
CF260 = 0.24
τfl
=
3.8 ns
CF280 = 0.11
τfl
=
3.2 ns
CF280 = 0.12
Features:
Features: • • • • • •
• • • • • •
High fluorescence yield High photostability Very hydrophilic Excellent water solubility Very little aggregation New dye with net charge of -1
High fluorescence yield High thermal and photostability Moderately hydrophilic Good solubility in polar solvents Cationic dye closely related to well-known Rhodamine 6G, perchlorate salt Supplied as mixture of three diastereomers
50
100
150
200
250
time of irradiation, min
300
fluorescence
350
300
400
500
600
700
wavelength, nm
800
900
1000
300
400
500
600
700
wavelength, nm
800
900
1000
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
with free COOH
765
646
1 mg 5 mg
AD 532-21 AD 532-25
with free COOH
694.2
594
1 mg 5 mg
AD 550-21 AD 550-25
NHS-ester
1081
743
1 mg 5 mg
AD 532-31 AD 532-35
NHS-ester
791.3
691
1 mg 5 mg
AD 550-31 AD 550-35
maleimide
1063
768
1 mg 5 mg
AD 532-41 AD 532-45
maleimide
816.4
716
1 mg 5 mg
AD 550-41 AD 550-45
biotin derivative
1357
956
1 mg 5 mg
AD 532-71 AD 532-75
biotin derivative
1004.7
904
1 mg 5 mg
AD 550-71 AD 550-75
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
28
absorbance
Cy3
0
fluorescence
absorbance
absorbance
ATTO 532
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
29
Fluorescent Labels
Fluorescent Labels ATTO 590
ATTO 565 Solvent: Water λabs
=
563 nm
εmax
=
λfl
Solvent: Water λabs
=
594 nm
1.2 x 10 M cm
εmax
=
1.2 x 105 M-1 cm-1
=
592 nm
λfl
=
624 nm
ηfl
=
90 %
CF260 = 0.34
ηfl
=
80 %
CF260 = 0.42
τfl
=
3.4 ns
CF280 = 0.16
τfl
=
3.7 ns
CF280 = 0.44
5
-1
-1
Features: • • • • • •
Features:
High fluorescence yield High thermal and photostability Good solubility in polar solvents Rhodamine dye related to well-known Rhodamine 101, perchlorate salt Supplied as mixture of two isomers with nearly identical properties Single isomer on request
• • • • • •
High fluorescence yield High thermal and photostability Good solubility in polar solvents New dye related to rhodamines, perchlorate salt Supplied as mixture of two isomers with nearly identical properties Single isomer on request HOOC
N
fluorescence
COOH
+ N
O ClO4
N
+ N
O
-
ClO4 300
400
500
600
700
wavelength, nm
800
900
1000
300
400
500
600
700
wavelength, nm
800
900
-
1000
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
with free COOH
611.0
511
1 mg 5 mg
AD 565-21 AD 565-25
with free COOH
691.2
591
1 mg 5 mg
AD 590-21 AD 590-25
NHS-ester
708.1
608
1 mg 5 mg
AD 565-31 AD 565-35
NHS-ester
788.3
688
1 mg 5 mg
AD 590-31 AD 590-35
maleimide
733.2
633
1 mg 5 mg
AD 565-41 AD 565-45
maleimide
813.3
713
1 mg 5 mg
AD 590-41 AD 590-45
biotin derivative
921.5
821
1 mg 5 mg
AD 565-71 AD 565-75
biotin derivative
1001.6
901
1 mg 5 mg
AD 590-71 AD 590-75
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
30
absorbance
absorbance
COOH
fluorescence
HOOC
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
31
Fluorescent Labels
Fluorescent Labels ATTO 610
ATTO 594 Solvent: Water λabs
=
601 nm
εmax
=
λfl
Solvent: Water λabs
=
615 nm
1.2 x 10 M cm
εmax
=
1.5 x 105 M-1 cm-1
=
627 nm
λfl
=
634 nm
ηfl
=
85 %
CF260 = 0.26
ηfl
=
70 %
CF260 = 0.02
τfl
=
3.5 ns
CF280 = 0.51
τfl
=
3.3 ns
CF280 = 0.05
5
-1
-1
Features:
fluorescence
High fluorescence yield High photostability Moderately hydrophilic Good solubility in all polar solvents Stable at pH 2 - 8 Cationic dye belonging to new class of carbopyronins, perchlorate salt
absorbance
• • • • • •
High fluorescence yield High photostability Very hydrophilic Excellent water solubility Very little aggregation New dye with net charge of -1
absorbance
• • • • • •
fluorescence
Features:
+ N
N ClO4
-
COOH 300
400
500
600
700
wavelength, nm
800
900
1000
400
500
600
700
wavelength, nm
800
900
1000
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
with free COOH
1137
806
1 mg 5 mg
AD 594-21 AD 594-25
with free COOH
491.0
391
1 mg 5 mg
AD 610-21 AD 610-25
NHS-ester
1389
903
1 mg 5 mg
AD 594-31 AD 594-35
NHS-ester
588.1
488
1 mg 5 mg
AD 610-31 AD 610-35
maleimide
1358
928
1 mg 5 mg
AD 594-41 AD 594-45
maleimide
613.1
513
1 mg 5 mg
AD 610-41 AD 610-45
biotin derivative
1456
1116
1 mg 5 mg
AD 594-71 AD 594-75
biotin derivative
801.4
701
1 mg 5 mg
AD 610-71 AD 610-75
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
32
300
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
33
Fluorescent Labels
Fluorescent Labels ATTO 620
ATTO 611X Solvent: Water λabs
=
611 nm
εmax
=
λfl
Solvent: Water λabs
=
619 nm
1.0 x 10 M cm
εmax
=
1.2 x 105 M-1 cm-1
=
681 nm
λfl
=
643 nm
ηfl
=
35 %
CF260 = 0.05
ηfl
=
50 %
CF260 = 0.05
τfl
=
2.5 ns
CF280 = 0.07
τfl
=
2.9 ns
CF280 = 0.07
5
-1
-1
Features:
BF4
-
COOH
300
400
500
600
700
wavelength, nm
800
900
1000
300
400
500
600
700
wavelength, nm
800
900
1000
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
with free COOH
494.4
407
1 mg 5 mg
AD 611X-21 AD 611X-25
with free COOH
612.1
512
1 mg 5 mg
AD 620-21 AD 620-25
NHS-ester
591.5
504
1 mg 5 mg
AD 611X-31 AD 611X-35
NHS-ester
709.2
609
1 mg 5 mg
AD 620-31 AD 620-35
maleimide
616.5
529
1 mg 5 mg
AD 611X-41 AD 611X-45
maleimide
734.3
634
1 mg 5 mg
AD 620-41 AD 620-45
biotin derivative
804.8
717
1 mg 5 mg
AD 611X-71 AD 611X-75
biotin derivative
922.6
822
1 mg 5 mg
AD 620-71 AD 620-75
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
34
High fluorescence yield High thermal and photostability Moderately hydrophilic Good solubility in all polar solvents Stable at pH 4 - 11 Cationic dye, perchlorate salt
absorbance
fluorescence
+ N
N
• • • • • •
fluorescence
Fluorescence yield unusually high in this wavelength region Large Stokes-shift High photostability Good solubility in polar solvents At pH > 5 reversible formation of colorless pseudobase Cationic dye, tetrafluoroborate salt
absorbance
• • • • • •
Features:
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
35
Fluorescent Labels
Fluorescent Labels ATTO 635
ATTO 633 Solvent: Water λabs
=
629 nm
εmax
=
λfl
Solvent: Water λabs
=
635 nm
1.3 x 10 M cm
εmax
=
1.2 x 105 M-1 cm-1
=
657 nm
λfl
=
659 nm
ηfl
=
64 %
CF260 = 0.05
ηfl
=
25 %
CF260 = 0.13
τfl
=
3.2 ns
CF280 = 0.06
τfl
=
1.9 ns
CF280 = 0.10
5
-1
-1
Features: • • • • • •
Features:
High fluorescence yield High thermal and photostability Moderately hydrophilic Good solubility in all polar solvents Stable at pH 4 - 11 Cationic dye, perchlorate salt
• • • • • •
High fluorescence yield High photostability Moderately hydrophilic Good solubility in all polar solvents Stable at pH 2 - 8 Cationic dye belonging to new class of carbopyronins, perchlorate salt
fluorescence
Cy5
absorbance
fluorescence
absorbance
absorbance
ATTO 633
+ N
N ClO4
0
10
20
30
40
time of irradiation, min
50
COOH
60
300
400
500
600
700
wavelength, nm
800
900
1000
300
400
500
600
700
wavelength, nm
800
900
1000
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
with free COOH
652.2
552
1 mg 5 mg
AD 633-21 AD 633-25
with free COOH
531.1
431
1 mg 5 mg
AD 635-21 AD 635-25
NHS-ester
749.3
649
1 mg 5 mg
AD 633-31 AD 633-35
NHS-ester
628.1
528
1 mg 5 mg
AD 635-31 AD 635-35
maleimide
774.3
674
1 mg 5 mg
AD 633-41 AD 633-45
maleimide
653.2
553
1 mg 5 mg
AD 635-41 AD 635-45
biotin derivative
962.7
862
1 mg 5 mg
AD 633-71 AD 633-75
biotin derivative
841.5
741
1 mg 5 mg
AD 635-71 AD 635-75
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
36
-
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
37
Fluorescent Labels
Fluorescent Labels ATTO 647
ATTO 637 Solvent: Water λabs
=
635 nm
εmax
=
λfl
Solvent: Water λabs
=
645 nm
1.2 x 10 M cm
εmax
=
1.2 x 105 M-1 cm-1
=
659 nm
λfl
=
669 nm
ηfl
=
25 %
CF260 = 0.05
ηfl
=
20 %
CF260 = 0.08
τfl
=
1.9 ns
CF280 = 0.02
τfl
=
2.3 ns
CF280 = 0.04
5
-1
-1
Features:
fluorescence 300
400
500
600
700
wavelength, nm
800
900
1000
300
400
500
600
700
wavelength, nm
800
900
1000
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
with free COOH
552
511
1 mg 5 mg
AD 637-21 AD 637-25
with free COOH
592
593
1 mg 5 mg
AD 647-21 AD 647-25
NHS-ester
838
608
1 mg 5 mg
AD 637-31 AD 637-35
NHS-ester
811
690
1 mg 5 mg
AD 647-31 AD 647-35
maleimide
716
633
1 mg 5 mg
AD 637-41 AD 637-45
maleimide
832
715
1 mg 5 mg
AD 647-41 AD 647-45
biotin derivative
929
822
1 mg 5 mg
AD 637-71 AD 637-75
biotin derivative
1219
903
1 mg 5 mg
AD 647-71 AD 647-75
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
38
High fluorescence yield High photostability Very hydrophilic Good solubility in all polar solvents Stable at pH 2 - 8 Zwitterionic dye
absorbance
• • • • • •
High fluorescence yield High photostability Very hydrophilic Good solubility in all polar solvents Stable at pH 2 - 8 Zwitterionic dye
absorbance
• • • • • •
fluorescence
Features:
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
39
Fluorescent Labels
Fluorescent Labels ATTO 655
ATTO 647N Solvent: Water λabs
=
644 nm
εmax
=
λfl
Solvent: Water λabs
=
663 nm
1.5 x 10 M cm
εmax
=
1.25 x 105 M-1 cm-1
=
669 nm
λfl
=
684 nm
ηfl
=
65 %
CF260 = 0.06
ηfl
=
30 %
CF260 = 0.24
τfl
=
3.4 ns
CF280 = 0.05
τfl
=
1.9 ns
CF280 = 0.08
5
-1
-1
Features: Extraordinarily high fluorescence yield at this wavelength High thermal and photostability Excellent ozone resistance Moderately hydrophilic Good solubility in polar solvents Stable at pH 4 - 11 Cationic dye, perchlorate salt, mixture of two isomers
• • • • • • •
10
20
30
40
time of irradiation, min
50
60
Cy5
0
300
400
500
600
700
wavelength, nm
800
900
1000
300
400
500
600
700
wavelength, nm
800
900
1000
10
20
30
40
time of irradiation, min
50
60
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
with free COOH
746.3
646
1 mg 5 mg
AD 647N-21 AD 647N-25
with free COOH
634
528
1 mg 5 mg
AD 655-21 AD 655-25
NHS-ester
843.4
743
1 mg 5 mg
AD 647N-31 AD 647N-35
NHS-ester
887
625
1 mg 5 mg
AD 655-31 AD 655-35
maleimide
868.5
768
1 mg 5 mg
AD 647N-41 AD 647N-45
maleimide
812
650
1 mg 5 mg
AD 655-41 AD 655-45
biotin derivative
1056.8
956
1 mg 5 mg
AD 647N-71 AD 647N-75
biotin derivative
1204
838
1 mg 5 mg
AD 655-71 AD 655-75
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
40
absorbance
fluorescence
absorbance
absorbance
Cy5
fluorescence
ATTO 655
ATTO 647N
0
High fluorescence yield Excellent thermal and photostability Excellent ozone resistance Electron transfer quenching of fluorescence by guanine, tryptophan, etc. Very hydrophilic Good solubility in all polar solvents Zwitterionic dye
absorbance
• • • • • • •
Features:
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
41
Fluorescent Labels
Fluorescent Labels ATTO 700
ATTO 680 Solvent: Water λabs
=
680 nm
εmax
=
λfl
Solvent: Water λabs
=
700 nm
1.25 x 10 M cm
εmax
=
1.2 x 105 M-1 cm-1
=
700 nm
λfl
=
719 nm
ηfl
=
30 %
CF260 = 0.30
ηfl
=
25 %
CF260 = 0.26
τfl
=
1.8 ns
CF280 = 0.17
τfl
=
1.5 ns
CF280 = 0.41
5
-1
-1
Features:
400
500
600
700
wavelength, nm
800
900
1000
High fluorescence yield Excellent thermal and photostability Electron transfer quenching of fluorescence by guanine, tryptophan, etc. Very hydrophilic Good solubility in all polar solvents Zwitterionic dye
absorbance
fluorescence 300
300
400
500
600
700
wavelength, nm
800
900
1000
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
with free COOH
631
526
1 mg 5 mg
AD 680-21 AD 680-25
with free COOH
575
566
1 mg 5 mg
AD 700-21 AD 700-25
NHS-ester
828
623
1 mg 5 mg
AD 680-31 AD 680-35
NHS-ester
837
663
1 mg 5 mg
AD 700-31 AD 700-35
maleimide
1024
648
1 mg 5 mg
AD 680-41 AD 680-45
maleimide
971
688
1 mg 5 mg
AD 700-41 AD 700-45
biotin derivative
1123
836
1 mg 5 mg
AD 680-71 AD 680-75
biotin derivative
973
876
1 mg 5 mg
AD 700-71 AD 700-75
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
42
• • • • • •
High fluorescence yield Excellent thermal and photostability Electron transfer quenching of fluorescence by guanine, tryptophan, etc. Very hydrophilic Good solubility in all polar solvents Zwitterionic dye
absorbance
• • • • • •
fluorescence
Features:
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
43
Fluorescent Labels
Fluorescent Labels ATTO 740
ATTO 725 Solvent: Water
Solvent: Water
λabs
=
729 nm
λabs
=
740 nm
εmax
=
1.2 x 105 M-1 cm-1
εmax
=
1.2 x 105 M-1 cm-1
λfl
=
752 nm
λfl
=
764 nm
ηfl
=
10 %
CF260 = 0.10
ηfl
=
10 %
CF260 = 0.11
τfl
=
0.5 ns
CF280 = 0.08
τfl
=
0.6 ns
CF280 = 0.10
Features:
Features: • • • • •
High thermal and photostability Excellent photostability Moderately hydrophilic Good solubility in polar solvents Cationic dye, perchlorate salt
• • • • •
High thermal and photostability Excellent photostability Moderately hydrophilic Good solubility in polar solvents Cationic dye, perchlorate salt
10
20
30
40
time of irradiation, min
50
absorbance
fluorescence
60
Cy5
0
300
400
500
600
700
wavelength, nm
800
900
1000
300
400
500
600
700
wavelength, nm
800
900
1000
10
20
30
40
time of irradiation, min
50
60
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
with free COOH
516.0
416
1 mg 5 mg
AD 725-21 AD 725-25
with free COOH
568.1
468
1 mg 5 mg
AD 740-21 AD 740-25
NHS-ester
613.1
513
1 mg 5 mg
AD 725-31 AD 725-35
NHS-ester
665.1
565
1 mg 5 mg
AD 740-31 AD 740-35
maleimide
638.1
538
1 mg 5 mg
AD 725-41 AD 725-45
maleimide
690.2
590
1 mg 5 mg
AD 740-41 AD 740-45
biotin derivative
826.5
726
1 mg 5 mg
AD 725-71 AD 725-75
biotin derivative
878.5
778
1 mg 5 mg
AD 740-71 AD 740-75
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
44
absorbance
Cy5
0
fluorescence
ATTO 740
absorbance
absorbance
ATTO 725
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
45
Fluorescence Quenchers
Fluorescence Quenchers ATTO 540Q Solvent: Water
Fluorescence resonance energy transfer (FRET) from an excited dye molecule (donor) to another nearby dye molecule (acceptor) leads to deactivation of the donor, i.e. it no longer fluoresces: Its fluorescence is quenched. The process of FRET depends, among other factors, on the absorption spectrum of the acceptor, as was discussed in some detail on p. 10-11. If the acceptor is fluorescent itself, it will emit light just the same, as if it had been excited directly (without utilisation of the donor). However, if the acceptor is non-fluorescent, it will merely accept excitation energy from the donor, yet not produce any fluorescence by its own. Such acceptors are called “fluorescence quenchers”.
λabs
=
542 nm
εmax
=
1.05 x 105 M-1 cm-1 CF260 = 0.22 CF280 = 0.24
Features: Fluorescence quenchers reduce the fluorescence intensity of the donor dye according to the formulas given on p. 10-11. The Förster-radius R0 is determined by the overlap between fluorescence spectrum of the donor and absorption spectrum of the acceptor (quencher). For efficient quenching the absorption region of the quencher must overlap well with the fluorescence spectrum of the donor.
• • • •
High thermal and photostability Moderately hydrophilic Good solubility in polar solvents Cationic rhodamine dye, perchlorate salt
Note: The fluorescence of dyes may be quenched also by mechanisms entirely different than FRET. For example, the fluorescence of ATTO 655, ATTO 680, and ATTO 700 is quenched very efficiently by guanosine, tryptophan and related compounds. This process is based on electron transfer and requires direct contact between excited dye molecule and quenching agent.
absorbance
ATTO-TEC provides quenchers covering most of the relevant visible spectrum. Their properties are outlined on p. 47-49. The Förster-radii R0 for combinations with fluorescent ATTO-labels as donors are presented in the table on p. 12-13.
300
400
500
600
700
wavelength, nm
800
900
1000
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
with free COOH
659.1
559
1 mg 5 mg
AD 540Q-21 AD 540Q-25
NHS-ester
756.2
656
1 mg 5 mg
AD 540Q-31 AD 540Q-35
maleimide
781.2
681
1 mg 5 mg
AD 540Q-41 AD 540Q-45
biotin derivative
969.6
869
1 mg 5 mg
AD 540Q-71 AD 540Q-75
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
46
47
Fluorescence Quenchers
Fluorescence Quenchers ATTO 612Q
ATTO 580Q Solvent: Water λabs
=
586 nm
εmax
=
1.1 x 10 M cm 5
-1
Solvent: Water
-1
615 nm
εmax
=
1.15 x 105 M-1 cm-1 CF260 = 0.35
CF280 = 0.13
CF280 = 0.57
Features: High thermal and photostability Moderately hydrophilic Good solubility in polar solvents Cationic dye related to rhodamines, perchlorate salt
absorbance
• • • •
absorbance
High thermal and photostability Moderately hydrophilic Good solubility in polar solvents Cationic dye related to rhodamines, perchlorate salt Supplied as mixture of three diastereomers
300
400
500
600
700
wavelength, nm
800
900
1000
300
400
500
600
700
wavelength, nm
800
900
1000
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
Modification
MW, g/mol
M+, g/mol
Unit
Order Code
with free COOH
795.3
695
1 mg 5 mg
AD 580Q-21 AD 580Q-25
with free COOH
791.3
691
1 mg 5 mg
AD 612Q-21 AD 612Q-25
NHS-ester
892.4
792
1 mg 5 mg
AD 580Q-31 AD 580Q-35
NHS-ester
888.4
788
1 mg 5 mg
AD 612Q-31 AD 612Q-35
maleimide
917.5
817
1 mg 5 mg
AD 580Q-41 AD 580Q-45
maleimide
913.4
813
1 mg 5 mg
AD 612Q-41 AD 612Q-45
biotin derivative
1105.8
1005
1 mg 5 mg
AD 580Q-71 AD 580Q-75
biotin derivative
1101.8
1001
1 mg 5 mg
AD 612Q-71 AD 612Q-75
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
48
=
CF260 = 0.36
Features: • • • • •
λabs
Other conjugates with, e.g. streptavidin, sec. antibodies, phalloidin etc. on request.
49
Large Stokes-Shift Dyes
Large Stokes-Shift Dyes
Dyes with Large Stokes-Shift On excitation of a dye molecule a reorientation of the π-electron system takes place. This occurs extremely fast (faster than picoseconds). Due to the new charge distribution about the dye molecule the surrounding solvent molecules also move towards new equilibrium positions. As a consequence the energy of the entire system (excited dye molecule plus solvent) is lowered quickly, and the photons emitted have a lower energy than those needed for excitation. In other words: The fluorescence occurs at longer wavelengths than the excitation. The wavelength difference between fluorescence maximum and the corresponding absorption maximum is called Stokes-shift. With typical dyes in aqueous solution the value of the Stokes-shift is 20 – 30 nm.
Optical Properties in Water Label
εmax ,
λabs ,
λfl ,
M-1 cm-1
nm
nm
nm
Stokes-Shift,
τfl ,
%
ns
Page
ATTO 390
24000
390
479
89
90
3.8
22
ATTO 425
45000
436
484
48
90
3.5
23
ATTO 465
75000
453
508
55
55
2.2
24
ATTO 611X
100000
611
681
70
35
2.5
34
On excitation of dyes with highly unsymmetrical π-electron systems the dipole moment may change drastically. The ensuing strong reorientation of solvent molecules leads to an unusually large Stokes-shift, in particular in polar solvents like water and ethanol. As the non-radiative decay of the excited state is also enhanced by the solvent reorientation, the fluorescence quantum yield of such compounds is severely reduced in aqueous solutions. However, there are a few exceptions to this rule: Coumarin derivatives like ATTO 390 and ATTO 425 show a remarkably large Stokes-shift of about 90 and 50 nm, respectively, and yet fluoresce with a quantum yield of 90 % in water (table p. 21).
ATTO 390
ATTO 425 O O
N
O
N
O
COOH
Even more remarkable are the dyes ATTO 465 and ATTO 611X. In spite of their symmetrical structure they have large Stokes-shifts of 55 and 70 nm, respectively. The fluorescence quantum yield of 35 % in aqueous solution, measured for ATTO 611X, is among the highest found for dyes emitting in the far red region.
+ N
O
ATTO 611X
NH2
+ N
N
ClO4
O
COOH
ATTO 465
H2N
50
ηfl ,
-
COOH
BF4
-
COOH
51
Customized Dyes and Services
Customized Dyes and Services
Customized Labels and Products In addition to the products described in this catalogue ATTO-TEC is pleased to offer on request dyes and labels taylored to the special needs of its customers. The following examples may illustrate the possibilities.
Conjugates Fluorescent conjugates of ATTO-dyes with streptavidin, phalloidin, sec. antibodies, and many other proteins are prepared on request.
Derivatives of ATTO-Labels Special Dyes Linker In most ATTO-labels the reactive group (NHS-ester etc.) is connected with the fluorophore by a linker consisting of a 4-atom flexible chain. For many applications this has proven to be very suitable and practical. However, if your experiment requires a linker of different length, rigidity, or other special feature, - most likely we are able to provide it.
Reactive group N-hydroxysuccinimide (NHS) ester and maleimide are the most common reactive groups for coupling to amine and thiol, respectively. However, for other substrate functionalities it is necessary that the label carries an entirely different reactive group: ATTO-TEC can provide amine, hydrazide (for coupling to aldehyde), and many others.
Bichromophoric Dyes If two fluorescent chromophores are connected by a linker, energy transfer (FRET) may occur intramolecularly. Thereby the fluorescence of the short-wavelength chromophore is quenched, and fluorescence from the long-wavelength chromophore is observed exclusively. The absorption spectrum of such bichromophoric dye resembles the superposition of the individual spectra. Therefore the dye absorbs very well in a wavelength range considerably wider than in case of a single chromophore, its fluorescence can be excited better with a broad-band light source. Although bichromophoric dyes are by necessity of larger size than normal labels, they may have an advantage in certain applications. ATTO-TEC will supply such dyes on request.
pH-Sensitive Dyes Solubility, Charges On customer request ATTO-dyes can be rendered very hydrophobic or else very hydrophilic and thus become compatible with the corresponding solvents, surfaces, or biochemical environments. Cell permeability can be influenced in broad limits. Also dyes may be shielded by a dendrimeric shell. The electrical charge can be adapted to achieve the desired interaction with a biomolecule or simply to obtain a special migration behaviour in electrophoresis.
52
ATTO-TEC has the capacity to supply various dyes, whose fluorescence efficiency depends strongly on the acidity of the solution - or environment, generally speaking. Depending on the particular molecular structure, such dye will fluoresce in acidic (low pH) or in basic (high pH) environment. The absorption spectrum also may change with pH. Customers are welcome to ask for details.
53
Customized Dyes and Services
Customized Dyes and Services
Triplet Dyes On optical excitation of a dye molecule there is always a certain probability that the molecule is converted to the triplet state, a relatively long-lived excited state of the dye molecule. The occurrence of this state is frequently not desirable, as it promotes destruction (bleaching) of the dye. Nevertheless dyes with high triplet yield find application in photochemistry, photodynamic therapy etc. They are efficient sensitizers for the conversion of molecular oxygen (air) into its highly reactive form (singulet oxygen). In addition to the acridine dyes ATTO 465 (p. 24) and ATTO 495 (p. 26), both absorbing below 500 nm, we supply on customer’s request triplet labels derived from Thiorhodamine. This dye, absorbing at 580 nm, can be provided as NHS-ester and as maleimide for coupling.
because it lacks the necessary reactive groups. However, ATTO-TEC supplies hydrophilic derivatives of this dye activated as NHS-ester or maleimide on request.
H
-
Cl + N
S
Methylene Blue blue
COOH
+ N
S
N
HCl
N
[red.] N
[ox.]
N
N S
N
leukobase colorless
Protease-active Dyes ATTO-TEC has developed a series of dye derivatives which become fluorescent only when activated by the corresponding enzyme (protease). These compounds, very useful for the determination of protease activity, are supplied on request.
Redox Dyes A dye, well-known in biochemical and medical research, is Methylene Blue. It has very interesting redox properties: The dye, normally deep blue in color, is converted by mild reducing agents to its so-called leuko-form, which is colorless. Since this reaction is reversible, the blue color reappears on oxidation, e.g. by oxygen (air). These interconversions can be catalyzed enzymatically. - Methylene Blue as such cannot be coupled to biomolecules,
54
55
Labeling Procedures
Labeling Procedures
Recommended Procedures for Labeling
Procedure
Introduction
Dissolve 2 - 20 mg of protein in 1 ml of sodium bicarbonate buffer. Protein or peptide solutions must be free of any amine-containing substances such as Tris, free amino acids or ammonium ions. Antibodies that have been previously dissolved in buffers containing amines can be dialyzed against 10 - 20 mM phosphate-buffered saline (PBS), and the desired pH 8.3 for the labeling reaction can be obtained by adding 0.1 ml of 1 M sodium bicarbonate solution for each ml of antibody solution. The presence of low concentrations of sodium azide (< 3 mM) will not interfere with the labeling reaction.
ATTO-TEC offers a large variety of high-quality dyes for labeling amino and thiol groups. ATTO reactive dyes cover the spectral region from 350 nm in the UV to 750 nm in the NIR. The most commonly used amine-reactive reagents are N-hydroxysuccinimidyl(NHS)-esters. NHS-esters readily react with amine modified oligonucleotides or amino groups of proteins, i.e. the ε-amino groups of lysines or the amine terminus, forming a chemically stable amide bond between the dye and the protein or oligo. However, the amino group ought to be unprotonated to be reactive. Therefore the pH of the solution must be increased sufficiently to obtain a high concentration of unprotonated amino groups. On the other hand, the NHS-ester also reacts with the hydroxyl ions in the solution to yield free dye, which is no longer reactive. As the rate of this hydrolysis increases with the concentration of hydroxyl ions, the pH should be kept as low as possible. Buffering the solution at pH 8.3 has been found to be a good compromise between the contradicting requirements. Isothiocyanates also react with amino groups. However, in general the resulting thiourea compound is less stable and deteriorates over time. Sulfonyl chlorides are another group of amine-reactive compounds forming very stable sulfonamides, yet are more difficult to work with. Therefore NHS-esters are the preferred amine-reactive reagents for protein- or oligo-conjugation. For the labeling of thiol groups the most popular and commonly used reactive reagents are maleimides. ATTO maleimides react with thiol groups of proteins to form a stable thio-ether bond. Unlike the labeling of amino groups, thiol modifications generally take place at near neutral pH. Since most amino groups show very little reactivity at pH 7, thiol groups can be selectively labeled in the presence of amines.
Labeling Proteins with Amine-Reactive ATTO-Labels (NHS-Esters) We recommend using 0.1 - 0.2 M sodium bicarbonate buffer of pH 8.3 for labeling proteins. Number and surface position of amino groups vary considerably among different proteins. Therefore it is advisable that different degrees of labeling (DOL) be tried in order to find the most satisfactory solution for the problem at hand.
56
Dissolve the amine-reactive dye in anhydrous, amine-free DMF or DMSO at 1.0 mg/ml. Due to the high quality of ATTO NHS-esters such solutions are stable for a long period of time. However, it may be difficult to avoid humidity entering a solution in continuous use. Hence it is advisable to prepare, whenever possible, the dye solution immediately before starting the labeling reaction. To obtain a degree of labeling (DOL, dye-to-protein ratio) of 2 slowly add, while stirring, a threefold molar excess of reactive dye to the protein solution. Variations due to different reactivities of both the protein and the labeling reagent may occur. This may necessitate optimization of the dye to protein ratio used in the reaction in order to obtain the desired DOL. Incubate the reaction mixture for 1 hour at room temperature. However, in most cases the labeling reaction will be completed within 5-10 minutes. To increase the degree of labeling a higher ratio of NHS-ester to protein has to be used. Separation of the Conjugate from Free Dye Part of the applied dye NHS-ester will hydrolyze during the labeling reaction and must be removed from the labeled protein. We recommend using a Sephadex G-25 or equivalent gel filtration column (minimum of 1 cm diameter and 12 cm length; for very hydrophilic dyes a 20 cm column is preferable) for separation of protein from free dye. It is convenient to preequilibrate the column with phosphate buffered saline (PBS) or buffer of choice and to elute the protein using the same buffer. The first colored and fluorescent zone to elute will be the desired conjugate. A second colored and fluorescent, but slower moving zone contains the unlabeled free dye (hydrolyzed NHS-ester).
57
Labeling Procedures
If the antibody solution to be conjugated is very dilute, to avoid further dilution you may want to purify the conjugate by extensive dialysis. However, dialysis does not yield as efficient and rapid separation as gel filtration. To prevent denaturation of the conjugate after elution, add bovine serum albumin (BSA) or any other stabilizer of choice to a final concentration of 1 - 10 mg/ml.
Labeling Procedures
It follows for the degree of labeling, i.e. the average number of dye molecules coupled to a protein molecule: DOL = c(dye) / c(protein) and with the above relations:
DOL =
Storage of the Protein Conjugate In general, conjugates should be stored under the same conditions used for the unlabeled protein. For storage in solution at 4 °C, sodium azide (2 mM final concentration) can be added as a preservative. Removal of preservatives prior to use may be necessary to avoid inhibitory effects in applications in which conjugates are added to live cell specimens. The conjugate should be stable at 4 °C for several months. For long-term storage, divide the solution into small aliquots and freeze at -20 °C. Avoid repeated freezing and thawing. Protect from light. We recommend to centrifuge conjugate solutions in a micro-centrifuge before use. This step will remove any aggregates that may have formed during long-term storage. Determining the Degree of Labeling (DOL) The degree of labeling (DOL, dye-to-protein ratio) obtained by the above procedure can be determined by absorption spectroscopy making use of the Lambert-Beer law: Absorbance (A) = extinction coefficient (ε) × molar concentration × path length (d). Simply measure the UV-VIS spectrum of the conjugate solution as obtained after gel filtration in a quartz (UV-transparent) cell with 1 cm path length. You may need to dilute the solution, if it turns out to be too concentrated for a correct absorbance measurement. Determine the absorbance (Amax) at the absorption maximum (λabs) of the dye and the absorbance (A280) at 280 nm (absorption maximum of proteins). The concentration of bound dye is given by: c(dye) = Amax / εmax × d, where εmax is the extinction coefficient of the dye at the absorption maximum. The protein concentration is obtained in the same way from its absorbance at 280 nm. As all dyes show some absorption at 280 nm, the measured absorbance A280 must be corrected for the contribution of the dye. This is given by Amax × CF280. The values for the correction factor CF280 = ε280 / εmax are listed in the table on p.63. It follows for the absorbance of the protein itself: Aprot = A280 − Amax× CF280. Then the concentration of protein is: c(protein) = Aprot / εprot× d, where εprot is the extinction coefficient of the protein at 280 nm. 58
A max ⋅ ε prot A max / ε max = A prot / ε prot (A 280 − A max ⋅ CF280 ) ⋅ ε max
Note: The above relation is only valid if the extinction coefficient of the free dye εmax at the absorption maximum is the same as the extinction coefficient of the conjugated dye at this wavelength. Frequently this is not the case, so that the value calculated for DOL may be at fault by 20 % or more.
Labeling Proteins with Thiol-Reactive ATTO-Labels (Maleimides) ATTO maleimides readily react with thiol groups of proteins. The optimum pH for the modification of thiols with maleimides is pH 7.0 - 7.5. We recommend the reaction to be carried out in phosphate buffered saline (PBS). At this pH the thiol group is sufficiently nucleophilic to react with the maleimide, whereas the amino groups of the protein show only little reactivity at this pH due to a high degree of protonation. Procedure Dissolve the protein at 50 - 100 μM in PBS at pH 7.0 - 7.5. Reduction of disulfide bonds in the protein can be achieved by adding a tenfold molar excess of dithiothreitol (DTT) or other reducing agent. If DTT is used as a reducing agent, the excess has to be removed by dialysis prior to addition of the maleimide. This may not necessary with other reducing agents. We recommend to carry out the thiol modification in an inert atmosphere to prevent oxidation of the thiols, in particular if the protein has been treated with reagents such as dithiothreitol. In this case it may also be advisable to deoxygenate all buffers and solvents used for the thiol conjugation. Prepare a 1 - 10 mM stock solution of the ATTO maleimide in anhydrous DMSO or DMF. Note that such solutions are not stable for a long period of time. Hence we recommend to freshly prepare the dye solutions immediately prior to use. Add a 10 - 20 fold molar excess of reactive dye to the protein solution whilst stirring and incubate 2 hours at room temperature. 59
Labeling Procedures
To ensure that all reactive dye is consumed add an excess of a low molecular weight thiol, e.g. glutathione or mercaptoethanol.
Labeling Procedures
Recommended Buffers •
Separation of the Conjugate from Free Dye • To separate the thiol modified protein from unlabeled dye we recommend gel filtration using a Sephadex G-25 or equivalent gel filtration column (minimum of 1 cm diameter and 12 cm length; for very hydrophilic dyes a 20 cm column is preferable). Preequilibrate the column with phosphate buffered saline (PBS) or buffer of choice and elute the protein using the same buffer. The first colored and fluorescent zone to elute will be the thiol modified protein. A second colored and fluorescent, but slower moving zone contains the unlabeled free dye (hydrolyzed maleimide) and low-molecular-weight dye conjugate, i.e. the conjugate of excess maleimide with, e.g. mercaptoethanol.
Labeling Amine-Modified Oligonucleotides The oligonucleotide must be functionalized with an amino group at the 5‘-end. The procedure given below is for labeling of an amine-modified oligonucleotide of 18 to 24 bases in length and is valid for oligonucleotides containing a single amino group. For the success of the conjugation reaction the purity of the oligonucleotide is very important. It must be free of primary and secondary amines. Especially contaminations such as Tris, glycine and ammonium salts can inhibit the reaction. We therefore strongly recommend purification by extraction and precipitation of the sample prior to labeling: Purification of the Amine-Modified Oligonucleotide • • • • •
60
Dissolve 100 μg of the oligonucleotide in 100 μl demineralized water and extract three times with an equal amount of chloroform. Precipitate the oligonucleotide by adding 10 μl of 3 M sodium chloride and 250 μl of ethanol. Mix well and store at -20 °C for at least 30 minutes. Centrifuge the solution in a micro-centrifuge at about 12000 g for 30 minutes. Carefully remove the supernatant, rinse the pellet twice with small amounts of cold 70% ethanol and dry under vacuum. Finally dissolve the dry pellet in demineralized water to achieve a concentration of 25 μg/μl (4.2 mM for an 18-mer). This stock solution may be stored at -20 °C.
0.1 M sodium carbonate buffer (pH 9): Dissolve 0.5 nmol/μl sodium carbonate in demineralized water. 0.1 M sodium tetraborate buffer (pH 8.5): Dissolve 0.038 g of sodium tetraborate decahydrate for every ml of demineralized water. Adjust pH with hydrochloric acid to 8.5.
Either one of these buffers should be prepared as close as possible to the start of the labeling procedure. Small aliquots may be frozen immediately for long term storage. Avoid exposure to air for a long time as the uptake of carbon dioxide will lower the pH of the buffer. Procedure Allow the amine-reactive dye solution to reach room temperature before opening the vial. Dissolve the compound in anhydrous amine-free DMF or DMSO to achieve a concentration of 20 μg/μl. To 15 μl of this solution add 7 μl demineralized water, 75 μl buffer and 4 μl of 25 μg/μl stock solution of the amine-modified oligonucleotide. The reaction mixture may be a suspension rather than a clear solution. However, this does not affect the conjugation reaction. The mixture is stirred at room temperature for 3 to 6 hours. The yield of labeling will vary from 50 to 90 %. Longer incubation times do not necessarily result in greater labeling efficiency. Some loss of reactive dye is unavoidable due to hydrolysis of the NHS-ester. Note: The reaction may be scaled up or down as long as the concentrations of the components are maintained. However, significant alteration of the relative concentrations of the components may drastically reduce the labeling efficiency. Separation and Purification of the Conjugate We recommend the precipitation of the oligonucleotide with ethanol as the first step of purification. To achieve this, add 10 μl of 3 M sodium chloride and 250 μl of ethanol to the reaction mixture. Proceed as described under Purification of the amine-modified oligonucleotide. Note: Do not dry completely as the labeled oligonucleotide may become difficult to redissolve. The labeled oligonucleotide can be separated from unlabeled oligonucleotide and free dye by preparative gel electrophoresis or reversed-phase HPLC. 61
Labeling Procedures
Labeling Procedures
Purification by Gel Electrophoresis
Table: Optical properties of ATTO-Labels
For purification by gel electrophoresis use a 0.2 mm thick polyacrylamide slab gel with the following concentrations:
MW, g/mol NHS
Mal.
λabs, nm
ATTO 390
440
465
390
ATTO 425
498
523
ATTO 465
493
Suspend the residue from the ethanol precipitation in 200 μl of 50 % formamide. Heat to 55 °C and incubate for 5 minutes. This will disrupt any secondary structure. Load the warmed sample onto the gel and load an adjacent well with 50 % formamide containing 0.05 % Bromophenol Blue as indicator. The indicator has approximately the same Rf value (will migrate with approximately the same rate) as the oligonucleotide. Run the gel until the Bromophenol Blue has reached two thirds of the way down the gel. Remove the gels from the plates and place on Saran WrapTM. Lay the gel on a fluorescent TLC plate. Illuminate with UV-light at 366 nm. The band which shows fluorescence is the labeled oligonucleotide. Cut out the fluorescent band and purify using the crush and soak method or other suitable techniques. Fore more details, please refer to Sambrook J., Fritsch E.F. and Maniatis, T., Molecular cloning: A Laboratory Manual, Second Edition, Cold Spring Harbour Laboratory (1989).
ATTO 488
Purification by HPLC
< 25 bases 25 - 40 bases 40 - 100 bases
19 % polyacrylamide 15 % polyacrylamide 12 % polyacrylamide
For HPLC purification you may use a standard analytical RP-C18 (4.6 x 250 mm) column. Dissolve the residue from the ethanol precipitation in 0.1 M TEAA (triethylammonium acetate), load onto the column and run a linear solvent gradient of 0 - 75 % acetonitrile in 0.1 M TEAA with an increase of acetonitrile of 2 % per minute. This gradient should be adjusted for very hydrophobic labeled oligonucleotides up to 3 % per minute. For more hydrophilic dyes you should run a slower gradient of about 1 % per minute. In all cases the unlabeled oligonucleotide will migrate fastest followed by the labeled oligonucleotide and finally the free dye. For more details, please refer to Oliver R.W.A., HPLC of Macromolecules: A Practical Approach, IRL Press (1989).
62
Dye
εmax, M
CF260
CF280
2.4 x 104
0.52
0.08
436
4.5 x 10
4
0.27
0.23
518
453
7.5 x 10
4
1.12
0.54
981
1067
501
9.0 x 10
4
0.25
0.10
ATTO 495
549
574
495
8.0 x 10
4
0.57
0.39
ATTO 520
564
589
516
1.1 x 105
0.40
0.40
ATTO 532
1081
1063
532
1.15 x 10
5
0.22
0.11
ATTO 540Q
756
781
542
1.05 x 10
5
ATTO 550
791
816
554
ATTO 565
708
733
ATTO 580Q
892
ATTO 590
788
ATTO 594
1389
ATTO 610
-1
cm-1
0.22
0.24
1.2 x 10
5
0.24
0.12
563
1.2 x 10
5
0.34
0.16
917
586
1.1 x 10
5
0.36
0.13
813
594
1.2 x 105
0.42
0.44
1358
601
1.2 x 10
5
0.26
0.51
588
613
615
1.5 x 10
5
0.02
0.05
ATTO 611X
604
629
611
1.0 x 10
5
0.05
0.07
ATTO 612Q
888
913
615
1.15 x 10
0.35
0.57
ATTO 620
709
734
619
1.2 x 105
0.05
0.07
ATTO 633
749
774
629
1.3 x 10
5
0.05
0.06
ATTO 635
628
653
635
1.2 x 10
5
0.13
0.10
ATTO 637
838
716
635
1.2 x 10
5
0.05
0.02
ATTO 647
811
832
645
1.2 x 10
5
0.08
0.04
5
5
ATTO 647N
843
868
644
1.5 x 10
0.06
0.05
ATTO 655
887
812
663
1.25 x 105
0.24
0.08
ATTO 680
828
1024
680
1.25 x 10
ATTO 700
837
971
700
ATTO 725
613
638
ATTO 740
665
690
0.30
0.17
1.2 x 10
5
0.26
0.41
729
1.2 x 10
5
0.10
0.08
740
1.2 x 10
5
0.11
0.10
5
63
Labeled Nucleotides
Labeled Nucleotides
In cooperation with Jena Bioscience ATTO-TEC offers fluorescence-labeled nucleotides.
NH2 N O
Order code:
Features: • • •
EDA-AppNHp (EDAAMPPNP)
Labeling at different positions with spacers of different lengths. Labels that cover the entire visible spectrum. Extraordinary properties (e.g., good water solubility, high signal intensity, chemical and photochemical stability)
All nucleotides are available with the following ATTO-dyes. They are supplied as ready-to-use 1 mM aqueous solutions in units of 10 to 30 μl depending on the particular nucleotide and/or label. To create the applicable order code, please replace the xxx in the order codes below by the ATTO-dye number.
N6-(6-Amino)hexyl-ATP
HO
NU-810-xxx
P O
O
P
O
O
P O
O
O
O HN
EDA-ADP
NH2 N
Order code: O
NU-802-xxx
HO
P
N
O O
P
N
O
N
O
O O
O
2 Na
N
H
NU-805-xxx
O HO
O
P
O
P
O
N
O O
O
P
O
HN
O
EDA-ATP
H N N
O HO
O
P
O
P
O
O
P
O
O
O
NU-808-xxx
N
N
O
NH2 N
Order code:
NH
Order code:
HO
N
P
O O
O
P
P
O
N
O
O O
3 Na
O
N
N
O O
O
O OH
3 Na
ATTO-Label
HN
OH
OH
ATTO-Label
NU-835-xxx
O
N
O
3 Na
N6-(6-Amino)hexyl-dATP
ATTO-Label
HN
NH N
N
O
H
O
Order code:
O
3 Na
H N
ATTO-Label
O H N
N
N
O
H
O HN
8-[(6-Amino)hexyl]-aminoATP
NH2 N N H
Order code: NU-807-xxx
H N
ATTO-Label
O HO
P O
O O
P O 3 Na
O O
P
O
N
N
γ-(6-Aminohexyl)-ATP Order code:
O
O OH
HN
N
OH
NU-833-xxx
NH2
ATTO-Label N
HN O O
P O
O O
P O 3 Na
64
ATTO-Label
N
O O
P
O
N N
O
O OH
OH
65
Labeled Nucleotides
8-[(6-Amino)hexyl]-aminoadenosine-2‘,5‘-bisphosphate
Labeled Nucleotides
H N
ATTO-Label
NH2 N N H
N
O HO
Order code:
P
O
N
γ-(6-Aminohexyl)-GTP
ATTO-Label HN
Order code:
O
NU-834-xxx
N
N
O
O
O
NU-812-xxx
O
OH 2 Na
O
HO
O O
P
O
O
P
P
O
NH2 N N H P
O
N
N
O HO
Order code:
O
O OH
N O HO
N
P
O O
O
P
P
O
O P
NH2
O O
3 Na
HO
N
O
O
2 Na
NH
N
O O
O
O
NU-811-xxx
OH O
Order code: NU-820-xxx
NH2
N
O
EDA-GTP
H N
ATTO-Label
P
3 Na
O
8-[(6-Amino)hexyl]-aminoadenosine-3‘,5‘-bisphosphate
N
O O
NH
OH
O
H
O
O
HN
O
5-Propargylamino-dCTP
ATTO-Label
EDA-m7-GTP
O
Order code: NU-809-xxx HO
P
O O
O
P
N
O O
O
P
O
N
NU-824-xxx
O
O HO
P
O O
O
P
O O
O
P
O
N
NH2
O
O O
2 Na
OH
NH
N
O 3 Na
5-Propargylamino-CTP
O
O
CH3
Order code:
NH O
ATTO-Label
HN
NH
O
H
ATTO-Label
O HN
NH O
Order code:
NH
NU-831-xxx O HO
P O
O O
P
N
O O
O
P
O
O 3 Na
EDA-m -GDP
CH3
OH
NU-827-xxx
HN O
N
Order code:
O
OH
O
7
O HO
P O
P
NH
N
O O
O
ATTO-Label
N
NH2
O
O Na
O
O
H
O HN
HN
66
ATTO-Label
67
Labeled Nucleotides
8-[(6-Amino)hexyl]-aminoGMP
Labeled Nucleotides
H N
ATTO-Label
N N H
Order code:
P
O
NH2
N
ATTO-Label N H
NH
NU-825-xxx
O HO
O
O Na
N H
NU-832-xxx
NH
N
NU-826-xxx
O HO
8-[(6-Amino)hexyl]-aminoGTP NU-830-xxx
O P
O O
O
P
O
O
N O
O
O
OH
ATTO-Labels:
O
P
O
NH
N
O O
NH2
N
O
O OH
OH
3 Na
Aminoallyl-dUTP
ATTO-Label N H
O
Order code:
ATTO 390
ATTO 425
ATTO 465
ATTO 488
ATTO 495
ATTO 532
ATTO 540Q ATTO 550
ATTO 565
ATTO 580Q
ATTO 590
ATTO 594
ATTO 610
ATTO 611X
ATTO 612Q
ATTO 620
ATTO 633
ATTO 635
ATTO 637
ATTO 647N
ATTO 655
ATTO 680
ATTO 700
ATTO 725
ATTO 740
NH
NU-803-xxx
O HO
P
O O
O
P
N
O O
O
P
O
Aminoallyl-UTP
O
O
O OH
3 Na ATTO-Label N H
O
Order code:
NH
NU-821-xxx
O HO
P O
O O
P
N
O O
O
P
O
O
O
O 3 Na
68
O H2 C P
O
N H HO
P
Na
N
Order code:
O O
OH
H N
ATTO-Label
P
3 Na
O
O NH
O
O
OH
Order code: NH2
N
O
O
N H
O O
O
N O
ATTO-Label
O P
O
Aminoallyl-dUpCpp
O N
Order code:
O H2 C P
2 Na
H N
ATTO-Label
P
OH
OH
O
Order code:
NH
N
O HO
NU-829-xxx
8-[(6-Amino)hexyl]-aminocGMP
Aminoallyl-dUpCp
O
OH
OH
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Synthesis and Labeling of DNA
Picture Gallery
ATTO-TEC offers a full service for synthesis and labeling of oligonucleotides. The oligonucleotides are purified via HPLC twice. Failure sequences are removed during the first HPLC; the second HPLC is performed to remove unlabeled oligonucleotide as well as excess of dye. As a result a labeled oligonucleotide of excellent quality is obtained. The oligonucleotide can be labeled either at the 3‘ or the 5‘ end with the following dyes.
ATTO 390
ATTO 425
ATTO 465
ATTO 495
ATTO 520
ATTO 540Q
ATTO 550
ATTO 565
ATTO 580Q
ATTO 590
ATTO 594
ATTO 610
ATTO 611X
ATTO 620
ATTO 633
ATTO 635
ATTO 647N
ATTO 655
ATTO 680
ATTO 700
ATTO 725
ATTO 740
Tubulin (PtK2 - Male Rat Kangaroo Kidney Epithelial Cells) Tubulin mouse IgG primary antibody bound to tubulin. Immunostaining with ATTO 532 labeled sheep anti-mouse IgG secondary antibody.
Actin (PtK2 - Male Rat Kangaroo Kidney Epithelial Cells) ATTO 532 labeled phalloidin bound to actin.
All labeled oligonucleotides are supplied as aqueous solutions in PBS buffer pH 7.4.
Rat stomach: Actin stained with mouse anti-smooth muscle α-actin antibody and ATTO 488 anti-mouse IgG (green). Cytokeratin stained with polyclonal rabbit anti-cytokeratin and ATTO 647N antirabbit IgG (red).
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71
Synthesis Picture Gallery and Labeling of DNA
Picture Gallery
HUVEC: Vimentin/ATTO 532; E-Cadherin/ATTO 655 and DAPI
Imaging the spatial order of colloidal nanoparticles (a) Confocal image, (b) corresponding STED image. The silica nanoparticles feature a ATTO 532 fluorescent core and a non-fluorescent shell. Only the STED image (b) reveals grain boundaries, defects and dislocations in the semi-crystalline nanoparticle formation.
Revealing the nanopattern of the SNARE protein SNAP-25 on the plasma membrane of a mammalian cell. Confocal versus STED image of the antibody-tagged proteins. The secondary antibody was labeled with ATTO 532-NHS. The encircled areas show linearly deconvolved data. STED microscopy provides a substantial leap forward in the imaging of protein selfassembly.
72
HUVEC: Inhibitor apoptosis protein/ ATTO 550; E-Cadherin/ATTO 655 and DAPI
HUVEC: alpha-Tubulin/ATTO 532; E-Cadherin/ATTO 655 and DAPI
73
List of Abbreviations
Acknowledgments
Abbreviation ATTO-TEC GmbH is deeply grateful to the following individuals and companies for their contribution to this catalogue by providing fascinating images.
λ
wavelength
λabs
longest-wavelength absorption maximum
εmax
molar extinction coefficient at the longest-wavelength absorption maximum
ε260
molar extinction coefficient at λ = 260 nm
ε280
molar extinction coefficient at λ = 280 nm
• Prof. Dr. Peter Friedl and co-workers, Department of Organic Chemistry and Biochemistry, TU Darmstadt, Germany
CF260
CF260 = ε260/εmax. Correction factor used in the determination of degree of labeling (DOL) in case of dye-DNA conjugates.
• Sigma-Aldrich Production GmbH, Buchs, Switzerland
CF280
CF280 = ε280/εmax. Correction factor used in the determination of degree of labeling (DOL) in case of dye-protein conjugates.
λfl
fluorescence maximum
ηfl
fluorescence quantum yield
τfl
fluorescence decay time
τ0
natural (radiative) decay time
MW
molecular weight
M
molecular weight of dye cation (HPLC-MS)
+
MH
+
• Prof. Dr. Stefan Hell and co-workers, Department of NanoBiophotonics, MPI for Biophysical Chemistry, Göttingen, Germany
molecular weight of protonated dye (HPLC-MS)
DOL
degree of labeling
Trademarks:
HUVEC
human umbilical vein endothelial cells
Alexa 488, Alexa 532, Alexa 594, Alexa 633, Alexa 647 and Texas Red are trademarks of Invitrogen Corporation.
DAPI
4‘,6-diamidino-2-phenylindole
FITC
fluoresceinisothiocyanate
TAMRA
6-carboxytetramethylrhodamine
FAM
6-carboxyfluorescein
TET
tetrachloro-6-carboxyfluorescein
JOE
2,7-dimethoxy-4,5-dichloro-6carboxyfluorescein
HEX
hexachloro-6-carboxyfluorescein
ROX
6-carboxy-X-rhodamine
74
Cy3, Cy3.5, Cy5 and Cy5.5 are trademarks of GE Healthcare Group Companies. FAM, JOE, TET, HEX, ROX and TAMRA are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries.
Copyright © 2007 ATTO-TEC GmbH. All rights reserved.
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catalogue 2007/2008 Am Eichenhang 50 D-57076 Siegen Germany Phone: +49(0)-271-2 38 53-0 Fax: +49(0)-271-2 38 53-11 E-mail:
[email protected] http: www.atto-tec.com
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ATTO-TEC Fluorescent Labels and Dyes
ATTO-TEC GmbH
